title: Comparison of next-generation sequencing and clone-based sequencing in analysis of hepatitis B virus reverse transcriptase quasispecies heterogeneity.
abstract: We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = -Σi(pilnpi)/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.
authors.affiliation: Department of Infectious Diseases, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Gong
authors.forename: Yue
authors.initials: Y
authors.lastname: Han
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Pei
authors.initials: P
authors.lastname: Hao
authors.forename: Jia
authors.initials: J
authors.lastname: Sheng
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Li
authors.forename: De-Min
authors.initials: DM
authors.lastname: Yu
authors.forename: Qi-Ming
authors.initials: QM
authors.lastname: Gong
authors.forename: Fei
authors.initials: F
authors.lastname: Tian
authors.forename: Xiao-Kui
authors.initials: XK
authors.lastname: Guo
authors.forename: Xin-Xin
authors.initials: XX
authors.lastname: Zhang
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D016000:Cluster Analysis; D019295:Computational Biology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D014644:Genetic Variation; D006515:Hepatitis B virus / Q000145:classification* / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008297:Male; D010802:Phylogeny; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D017385:Sequence Homology; D044967:Serum / Q000821:virology
chemical_list: D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase